Intrinsic bent DNA is an alternative conformation of DNA molecule with phased tracts of adenine and thymine, repeated in each 10 base pairs, or multiple of them. Differently from flexibility, intrinsic bending sites permit a DNA curvature in particular regions as replications origins and promoters. The element involved in the ori-β replication origin in Drosophila melanosgaster third chromosome amplicon, the Amplification Control Element, ACE3, has been described elsewhere and was confirmed by several functional analyses. However, structural features in this locus are not described yet. The aim of our work is to map intrinsic bent DNA sites in the third chromosome amplicon segment that includes the ACE3, the S18 gene, the main replication origin ori-β and a partial sequence of the S15 gene. The analysis was performed using the electrophoretic behavior and computational analysis of the 7669 bp sequence. The helical parameters ENDS ratio, AT%, DG values and Roll angle were obtained using Map15a software. The ACE3 element with 315 bp and the ori-β segment was isolated with the multiple restriction enzymes digestion. These restriction fragments were separated in agarose (AGA) and polyacrylamide gels (PA) electrophoresis with and without ethidium bromide. Whereas AGA gels were used as the molecular weight control, PA gels detect fragments with mobility alteration, and PA gels with ethidium bromide confirmed the intrinsic curvature within the other conformations. The fragment's mobility pattern was obtained by calculating the R value (ratio between the apparent and the real fragment size in gels). R values ≥ 1.10 indicate mobility decrease and < 0.90 indicate faster mobility fragments. The 315 bp fragment which comprises the ACE3 region presented a significant R value, 1.25. The fragments that include the ori-β didn't show alteration in their mobility. The 7669 bp segment showed a 81% of A/T rate, low DG values, and a higher ENDS ratio value peak, 1.45 (2940 bp position) co-localized with the ACE3 segment, in which was detected a negative Roll angle value. These results add data about the structural features in the ACE3 of D. melanogaster, caring out future analyses on the possible participation of these bent sites in the amplification control process.